D-GL conjugate therapy

ABSTRACT

Receptor blocking technology using proteins conjugated to polymers of D-glutamic acid and D-lysine for treatment of antibody-mediated autoimmune disease, membrane and tumor disorders is disclosed.

This application is a division of application Ser. No. 07/254,597 filed,Oct. 7, 1988 now U.S. Pat. No. 4,950,469 which is a division ofapplication Ser. No. 869,393 filed Mar. 30, 1986 and now abandoned.

BACKGROUND OF THE INVENTION

D-GL (D-glutamic acid:D-lysine copolymer) conjugates have been describedat considerable length and various applications of D-GL conjugates ofvarious immunologically reactive species have been reported. Thefollowing U.S. Pat. Nos. describe in considerable detail the chemistryof D-GL and method of forming conjugates thereof: 4,191,668; 4,220,565;4,222,907; 4,253,995; 4,253,995; 4,276,206; and 4,388,441. Thetechnology underlying the invention is found in any of several treatisesand reports of current advances, including, for example: METHODS INENZYMOLOGY, Academic Press, New York, e.g. in Volume 58, Cell Cultureand Volume 68, Recombinant DNA; CELL BIOLOGY, Academic Press, New York(3 volumes); METHODS IN MOLECULAR BIOLOGY, Humana Press, Clifton, NewJersey; ADVANCES IN IMMUNOPHARMACOLOGY, Pergamon Press, New York;ADVANCES IN ALLERGOLOGY AND IMMUNOLOGY, Pergamon Press, New York, and inthe current technical and patent literature.

The immunochemistry of D-GL conjugates is discussed and specificexamples of immunochemical reaction, conjugation, and applications indiagnosis and treatment are discussed in the literature. See, forexample, the above listed U.S. Pat. Nos. and the following publications.Katz, David H.; Davie, J. M.; Paul, W. E.; Benacerraf, Baruj J. Exp.Mid. 1971 134(1) :201-223; Nossal, J. V.; Davie, J. M.; Paul, W. E.;Katz, David H.; Benacerraf, Baruj J. Exp. Med. 1972 136(3) :426-438;Katz, David H.; Toshiyuki, H.; Benacerraf, Baruj J. Exp. Med. 1972136(6) :1404-1429; Nossal, J. V.; Pike, B. L.; Katz, David. H. J. Exp.Med. 1973 138(1) :312-317; Osborne, D. P.; Katz, David. H. J. Exp. Med.1973 137(4) :991-1007; Katz, David H.; Toshiyuki, H.; Benacerraf, BarujProc. Nat. Acad. Sci. USA 1973 70(10) :2776-2780; Toshiyuki, H.; Katz,David H. J. Exp. Med. 1974 139:1446-1463; Katz, David H.; Toshiyuki, H.;Benacerraf, Baruj J. Exp. Med. 1974 139:1464-1471; Katz, David H.;Benacerraf, Baruj Immunol. Tol. 1974, 189-201 and 249-281; J. Immunol.1974, 112(3) :1158-1163; Ault, K. A.; Unanue, E. R.; Katz, David H.Proc. Natl. Acad. Sci. U.S.A. 1974, 71(8) :3111-3114; Eshhar, z.;Benacerraf, B; Katz, David H. J. Immunol. 1975, 114(2) :872-876; Katz,David H.; Stechsulte, D. J.; Benacerraf, Baruj J. Allerg. Clin. Immunol.1975 55(6) :403-410; Bullock, W. W., Katz, David H., Benacerraf, BarujJ. Immunol. 1975 115 (1) :272-277;Mosier, D. E. Nature (London1975:257:141-3; Bitter-Suermann, D.; Hadding, U.; Schorlemmer, H. U.;Limbert, M.; Dierich, M.; Dukor, P.J. Immunol. 1975 115(2) :425-30;Chiorazzi, N; Eshhar, Z; Katz, David H. Proc. Natl. Acad. Sci. U.S.A.1976 73(6) :2091-5; Katz, David H.; Borel, Y. J. Immunol. 1978 120(6):1824-1827; Liu, Fu-Tong; Katz, David H. Proc. Natl. Acad. Sci. USA 197976(3) :1430-1434; Liu, Fu-Tong; Bogowitz, C. A.; Bargatze, M. Z; Katz,Lee R.; Katz, David H. J. Immunol. 1979 123(6) :2456-2465; Katz, DavidH. and Liu, Fu-Tong, ADVANCES IN ALLERGOLOGY AND IMMUNOLOGY, Ed.Oehling, A., Pergamon Press, New York (1980) pp. 51-59; Liu, Fu-Tong;Bargatze, R. F.; Katz, David H. J. Allerg. Clin. Immunol. 1980 66(4):322-326; Katz, David H. and Liu, Fu-Tong, ADVANCES INIMMUNOPHARMACOLOGY, Ed. Hadden, J., Pergamon Press, New York (1980) pp.277-284; Klinman, N. R.; Schrater, A. F.; Katz, David H.J. Immunol. 1981126(5) :1970-1973.

The published literature describes research into various immunochemicalphenomena in an effort to understand the chain of immunological eventsand diagnostic methods and processes for detecting immunologicalspecies, e.g. antigens, haptens or antibodies to antigens or haptens.

The ability of D-GL conjugates to induce tolerance to a variety ofantigens, haptens, nucleotides, nucleosides, etc. is described and theability of such conjugates to suppress antibody responses is mentioned,see, e.g., U.S. Pat. No. 4,191,668, David H. Katz, Mar. 4, 1980.

The underlying premise in prior art methods and approaches is ability ofselected D-GL-antigen conjugates to interfere with the induction ofantibody responses and thereby modify, reduce or prevent the productionof antibodies. In the process of studying antibody tolerance, it hasbeen discovered that D-GL conjugates of cell surface receptor bindingmolecules which bind by specific receptors on the surface of cells andmembranes stabilize such cell surface receptor binding molecules at thespecific site and modifies, slows or prevents ingestion and/or migrationof the receptor. This discovery has both therapeutic and diagnosticimplications. For example, it is a feature of this invention to use cellsurface bonding molecule-D-GL conjugates to permit imaging of membranes,tumors, organs, etc. and to treat the surfaces of cells, membranes,tumors and organs by stabilizing cell surface receptor binding moleculeswhich bind to receptor sites thereon. It is also a feature of thisinvention to treat membranes, tumors and the like by applying to thesurface thereof cell surface binding molecule--D-GL conjugates tomodify, slow or prevent the normal response of the cell surface receptorwhen coupled with a normal cell surface binding molecule.

SUMMARY OF THE INVENTION

The present invention encompasses the use of cell surface bindingmolecule--D-GL conjugates as direct therapeutic reagents in thetreatment of cells, membranes, organs, tumors and the like.

The present invention also encompasses the use of cell surface bindingmolecule--D-GL conjugates as image enhancing reagents in the location,identification, and diagnosis of diseases or disorders of cells,membranes, organs, tumors and the like.

The invention may be described as a method of treating a disease ordisorder of cells, membranes, tumors and the like which have surfacecell receptors, and includes such steps as: identifying a cell surfacereceptor binding molecule having determinants which bind specifically tothe cell surface receptors, forming a cell surface bindingmolecule--D-GL conjugate of the cell surface receptor binding moleculeand introducing such D-GL conjugates into the patient.

The invention may also be described as a method of imaging cells,membranes, organ, tumors and the like which have surface cell receptors,and includes such steps as: identifying a cell surface receptor bindingmolecule having determinants which bind specifically to the cellreceptors, forming a conjugate of the cell surface receptor bindingmolecule with D-GL, introducing such D-GL conjugate into the patient,associating an image forming moiety with the conjugate, andphotographically, electronically or otherwise imaging the cell,membrane, organ, tumor or the like.

The invention contemplates cell receptor blocking reagents comprisingD-GL conjugates of cell surface receptor binding molecules which bindspecifically to cell surface receptors.

The invention includes methods and reagents for treating diseases anddisorders of cells, membranes, organs, tumors and the like with cellsurface binding molecule--D-GL conjugate which include toxins, painsuppressants, antibiotics, cell growth inhibitors, and other therapeuticreagents as moieties thereon.

The invention includes reagents comprising D-GL conjugates of proteinsof the type produced by or in the course of an autoimmune response in apatient; e.g. synthetic duplicates of all or portions of such proteins,genetically engineered equivalents of such proteins, etc., or isolatesof such proteins or portions thereof.

The invention also includes reagents comprising D-GL conjugates ofproteins of the type produced by or in the course of a patient'sresponse to activation by an antigen; e.g. synthetic duplicates of allor portions of such proteins, genetically engineered equivalents of suchproteins, etc., or isolates of such proteins or portions thereof.

The invention contemplates the identification of a portein produced by apatient as a result of activation by an autoimmune response or to anxenogenous antigenic response and the injection of a D-GL conjugates ofsuch proteins as a therapeutic method in treatment of the disease withwhich the response is associated.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention comprises the introduction of selected conjugatesof the copolymer of D-glutamic acid and D-lysine referred to generallyas D-GL into the patient, animal or human, to interfere with or inhibitthe progression of a disease or disorder and/or to define or diagnosethe disease or disorder as to location, extent of the invasion in thepatient's body, type or characterization or otherwise to assist in thetreatment and/or diagnosis of the patient's diseases or disorders.

In the discussion which follows, some terms are used in a more generalsense than might be considered rigorous as a precise definition of theterm. "Cell surface receptor binding molecule", for example, is used,unless otherwise indicated or modified, to mean a biological orbiochemical species which acts as a single biological or physiologicalunit and which reacts with specific binding determinants upon the cellsof a membrane, tumor, or other tissue. In many instances, such units areindeed single molecules. In other instances, such units may comprisemore than one molecular entity, as the term molecular may be rigidlydefined in the pure chemical bonding sense. Antibodies, generally, aresingle molecules and are within the meaning of the term "cell surfacereceptor binding molecule" as used herein. The essential feature of acell surface receptor binding molecule as the term is used here, is thatit is unitary in its physiological characteristics an binds selectivelythrough specific determinants to cell surface receptors. The terms"reagent" and "moiety" are used in an interrelated sense, as follows,unless the context or modifiers indicate a different meaning. A"reagent" may be any chemical, biological or biochemical material whichexhibits therapeutic or imaging characteristics and which can be reactedwith, bound to, or associated with D-GL conjugates of this invention orto D-GL. A "moiety" may be any chemical, biological or biochemicaladduct or substituent to D-GL conjugates of this invention or to D-GL asa precurser to such conjugates which exhibits therapeutic or imagingcharacteristics. "Therapy" and derivatives thereof as used here mean anyfunction, phenomena, or treatment which is beneficial to the patient."Patients" may be animals or humans. Receptor cell blocking means, asthe term is used here, preventing reaction of the cell surface receptorin the way it would otherwise react when bound with a normal, unmodifiedcell surface receptor binding molecule; e.g., preventing or reducing themobility of the receptor, preventing or reducing the ingestion of thereceptor and/or the blocking cell surface receptor binding molecule, ormodifying the normal physiological function of the cell surfacereceptor.

The following examples will illustrate the procedures, methods,compositions, and, to some extent, the mechanisms of this invention. Theexamples are intended to teach the concepts of the invention and are notlimiting in any manner or degree as to the application, utility or scopeof the invention.

The binding of a cell surface receptor binding molecule havingdeterminants specific for cell surface receptor may trigger one or moreresponses by the cell and in the host. Such responses may includemigration of the receptor in the membrane, migration of the receptorinto the cell where it may be metabolized to trigger an intracellularmetabolic function, or to induce or suppress a group of cellularmetabolic functions, transmission of a pain signal to the brain,proliferation of cells, syntheses of immunogen, histamine, complementfactors, lymphokines, helper factors, suppressor factors or otherphysiologically active or activity inducing or suppressing substances.It has now been discovered that the presence of a cell surface bindingcell surface receptor binding molecule-D-GL conjugate modifies suchresponse. It is possible to block such responses in a negative manner,i.e. simply interfere with the normal response or the normal rate ofresponse, or to modify the response in a positive way, i.e. induce abeneficial response, such as pain reduction or prevention, promotinghealing, etc. By including an imaging moiety, the cell surface, tumor,etc. may be imaged for diagnostic purposes. All such reactions arereferred to here as "blocking " because all involve blocking the cellreceptor from binding with a cell surface binding cell surface receptorbinding molecule which would induce the normal response.

T-cells are characterized by the presence of cell surface receptorswhich are capable of specifically binding antigens, though the exactnature of these receptors is not fully understood. There is someevidence that the T-cell surface receptor consists of one or two chains,but this has not been established to the complete satisfaction of allimmunologists. The presence of cell surface binding molecules, antigens,on T-cells is manifest in the response of the T-cell vis-a-vis aparticular activation of the T-cell. It is generally recognized thatthere is an interdependence between the antigen and MHC (majorhistocompatibility complex), but universal agreement upon the nature andextent of this interdependence has ben been reached. It is sufficientfor present purposes to recognize that it is difficult and in mostinstances impossible to predict what, if any, change in response may beexpected from the reaction of T-cell surface receptors with changed cellsurface binding molecules. There is substantial agreement that lectin-and antigen- activated T-cells produce the lymphokine referred to asT-Cell Growth Factor (TCGF) and interleukin-2 (IL-2). TCGF is a protein(MW 12,000-17,000) whose activity allows the long-term proliferation ofT-Cells followed by interaction with antigen. TCGF has been implicatedin the maintenance of T cell malignancies (Gootenberg, J. E., it al J.Exp. Med. 1981 154:1403). Natural killer cell activity may also bestimulated by TCGF (Henney, C. S. et al, Nature 1981 291:335). TCGFexists naturally in only very small amounts; however, recent advances inrecombinant DNA technology have made it possible to obtain TCGF inamounts suitable for rather detailed studies and other laboratorypurposes. According to this invention, D-GL conjugates of TCGF,TCGF-D-GL conjugates are introduced, by injection for example, into thepatient where such TCGF-D-GL conjugates will specifically bind to andinhibit the proliferation of TCGF dependent tumor cells, such asleukemias and lymphomas. It is significant that the specific cellsurface binding molecule--D-GL conjugate, TCGF-D-GL, without any otherreagent moiety has therapeutic effect. Further, if a specific tumorexpresses TCGF receptors, the TCGF-D-GL conjugate will bind to thesereceptors, thus offering diagnostic applications and by interfering withnon-conjugated TCGF binding to the tumor expressed TCGF receptorsdifferentiating between tumor and non-tumor express TCGF receptors,differentially inhibiting proliferation of tumor cells and be oftherapeutic value. Tumor toxic moieties attached to the D-GL backbonebound via the TCGF-D-GL conjugates act directly upon the tumor and mayreduce or prevent the proliferation of tumor cells and may kill existingtumor cells, depending upon the specific tumor toxic moiety utilized.

TCGF expressing hybridoma have been described (tanaguchi, et al. Nature1983 302:305) and the sequence thereof reported. TCGF is coupled to D-GLby the methods described by Katz (U.S. Pat. Nos. 4,191,668 and4,276,206). The resulting conjugate is suspended in a physiologicallyacceptable solution, e.g. isotonic saline solution, and injectedperiodically, e.g. daily, in amounts of from a few nanograms to 100micrograms the most efficacious amount being determined by starting witha minimum injection and increasing the injection while observing thetumor growth pattern until the growth is arrested and/or reversed.Injections may be into the blood stream or peritoneal fluid orintroduced adjacent to the tumor to be treated, or into a body fluidstream which enters an organ with which the tumor is associated.

Myasthenia gravis is an autoimmune disease in which the patient formsautoantibodies against muscle-cell receptors which normally recognizeand bind acetylcholine--the neurotransmitter released from nerveendings. Muscle movement signals are blocked and muscular weaknessresults. In addition to antibodies against acetylcholine receptors,autoantibodies against striated muscle fibers and thymic tissue, and, onoccasion, other autoantibodies may be produced by the patient. If itwere possible to selective clock the binding of the autoantibodies toacetylcholine receptors, muscle movement signals could reach thesereceptors, thus interrupting the chain of muscle weakening and otherdebilitating effects of myasthenia gravis. Conjugates of D-GL andacetylcholine receptor, the gene for which has been cloned and isbecoming well defined, prepared by the method of Katz, supra, in asuitable physiologically acceptable carrier solution injected in amountsof from less than a nanogram to over 100 micrograms per day, on aperiodic basis such as daily, will suppress the autoimmune response ofthe body to acetylcholine receptor antibody, thus reducing the progressof myasthenia gravis. In some instances, where diagnosis is early, thistreatment may reverse the progress of the disease and allow the muscleto rebuild lost strength by regular use.

Rheumatic fever is also believed to involve an autoimmune response tostreptococcal antigens that are expressed by other tissues, especiallycardiac tissue. The disease causes inflammation of joints and cardiactissue, for which there is presently no specific therapy. D-GLconjugates of antigens which specifically bind anti-Strep A antibodies,if injected in the microgram or submicrogram range, may be expected tointerfere with the autoimmune response which is believed to beimplicated in rheumatic fever. As specific autoantigens are identifiedand isolated, D-GL conjugates thereof may also interfere with theinflammation response and halt or diminish the rate of progress of thedisease. Such D-GL conjugates may include inflammation reducingmoieties, antibiotics and other therapeutic moieties coupled throughD-GL and the antigen to bind to the membrane surface, thus concentratingthe therapeutic agent on the membrane surface.

Distinct cell surface cell surface receptor binding molecules areassociated with various tumors, and are expressed differentially at highconcentrations on the surface of some tumors. D-GL conjugates ofanti-tumor antibody will bind specifically to the tumor, interferingwith normal tumor growth. Therapeutic agents, e.g. radioactive atoms ortoxins, which could not be tolerated on a whole-body basis may becoupled through D-GL to the specifically binding antibody, thusdirecting the therapeutic agent to the tumor, concentrating theradiation, toxicity or other therapeutic effect on the tumor surfacewhere the therapeutic effect is maximized and the overall side reactionis minimized. Beta and soft gamma emitting radioisotopes which normallyhave little or no value in therapy may be used with great effect becausethey are on the surface of the tissue to be attacked. Since the D-GLconjugate will concentrate preferentially on the tumor surface and willotherwise be spread throughout the body, the whole-body radiation isminimal while effective levels of radiation are concentrated on thetumor surface.

I⁻¹³¹ labeled cell surface binding molecule--D-GL conjugate may be used,for example, in the treatment of tumors which respond to radiotherapy byforming a conjugate of I⁻¹³¹ labeled D-GL with the cell surface bindingmolecule which binds specifically to the tumor. The radiation of theI⁻¹³¹ decay acts directly and immediately upon the tumor with minimalradiation upon other organs. A number of procedures for labellingreagents with I⁻¹³¹ and other radiolabels are disclosed in HANDBOOK OFRADIOIMMUNOASSAY, Guy E. Abraham, Marcell Dekker, Inc., New York, 1977,the references cited therein and in the more recent literature. Otherradiolabels may, of course, be used to accomplish desired types, levelsand lengths of radiation therapy to a particular tumor.

In some instances, through only minor invasion of the body, the cellsurface binding molecule D-GL-therapeutic conjugate may be applieddirectly to or adjacent the tumor, thus permitting the direct and mostefficient binding of the therapeutic agent specifically to the tumorwith minimum migration through the body. This procedure may be carriedout, for example, by using X-ray viewing or other techniques to guidethe tip of a hypodermic syringe to the desired location and introducingthe cell surface binding molecule D-GL conjugate on or adjacent thetumor. Injection may, as in the above examples, be through the bloodcirculatory system.

In addition to the purely therapeutic applications referred to, thesize, shape and location of a tumor may be determined with considerableaccuracy using the technique described to attach a visualizing moietysuch as a radioactive or radiopaque to the backbone of the D-GL of thecell surface binding molecule--D-GL conjugate or to the moleculethereof, and directing the conjugate to the tumor.

INDUSTRIAL APPLICATION

The present invention has general application to therapy in diseases inwhich receptor blocking technology is involved, and to the use of use ofproteins conjugated to polymers of D-glutamic acid and D-lysine fortreatment of antibody-mediated autoimmune disease.

What is claimed is:
 1. A conjugate of D-GL and a cell surface bindingmolecule wherein the cell surface binding molecule is a toxin, painsuppressant, antibiotic or cell growth inhibitor.
 2. A method fortreating a patient for disease or disorder comprising administering tothe patient a conjugate of D-GL and a cell surface binding moleculewherein the cell surface binding molecule is a toxin, pain suppressant,antibiotic or cell growth inhibitor that is effective in treating thedisease or disorder.